Hyperchlosterolemia is known to be one of the prime risk factors for ischemic cardiovascular diseases such as arteriosclerosis. Cholesterol and other lipids are transported in body fluids by lipoproteins of varying density. The two lipoproteins carrying the majority of cholesterol in the blood are low-density lipoproteins (LDL) and high-density lipoproteins (HDL). The role of LDL is to transport cholesterol to peripheral cells outside the liver. LDL-receptors on a cell plasma membrane bind LDL and allow entry of cholesterol into the cell. HDL may scavenge cholesterol in the tissues for transport to the liver and eventual catabolism. LDL levels are positively correlated with the risk of coronary artery disease while HDL levels are negatively related, and the ratio of LDL-cholesterol to HDL-cholesterol has been reported to be the best predictor of coronary artery disease. Thus substances which effectuate mechanisms for lowering LDL-cholesterol may serve as effective antihypercholesterolemic agents.
Mevacor.RTM. (lovastatin; mevinolin) and ZOCOR.RTM. (simvastatin), now commercially available, are two of a group of very active antihypercholesterolemic agents that function by inhibiting the enzyme HMG-CoA reductase. Lovastatin and related compounds inhibit cholesterol synthesis by inhibiting the rate-limiting step in cellular cholesterol biosynthesis, namely the conversion of hydroxymethyl-glutarylcoenzyme A (HMG-CoA) into mevalonate by HMG-CoA reductase [3.7-9.12]. HMG-CoA reductase inhibitors act through cellular homeostatic mechanisms to increase LDL receptors with a consequent reduction in LDL-cholesterol and a resultant therapeutic antihypercholesterolemic effect. The HMG-CoA reductase inhibitors within this invention include, but are not limited to compactin (ML-236B), lovastatin, simvastatin, pravastatin, fluvastatin and mevastatin.
Many HMG-CoA reductase inhibitors are synthesized by microorganisms. The general biosynthetic pathway of the HMG-CoA reductase inhibitors of the present invention has been outlined by Moore et al., who showed that the biosynthesis of mevinolin (lovastatin) by Aspergillus terreus ATCC 20542 proceeds from acetate via a polyketide pathway (R. N. Moore et al., Biosynthesis of the hypocholesterolemic agent mevinolin by Aspergillus terreus. Determination of the origin of carbon, hydrogen, and oxygen atoms by .sup.13 C NMR and mass spectrometry. J. Amer. Chem. Soc., 1985, 107: 3694-3701). Endo and his coworkers demonstrated that similar biosynthetic pathways existed in Pencillium citrinum NRRL 8082 and Monascus ruber M-4681 (A. Y. Endo et al., Biosynthesis of ML-236B (compactin) and monacolin K., 1985, J. Antibiot., 38:444-448).
The recent commercial introduction of HMG-CoA reductase inhibitors has provided a need for high yielding processes for their production. Methods of improving process yield include, but are not limited to scaling up the process, improving the culture medium or, simplifying the isolation train. The present invention focuses on a method of increasing process yield wherein the increase in productivity is due to the use of a microorganism that produces increased levels of HMG-CoA reductase inhibitor.
It may be desirable to increase the biosynthesis of HMG-CoA reductase inhibitors at the level of gene expression. Such increases could be achieved by increasing the concentration in an HMG-CoA reductase inhibitor-producing microorganism of one or more of the enzymes or enzymatic activities in the biosynthetic pathway of the HMG-CoA reductase inhibitor. It may be particularly desirable to increase the concentration of a rate-limiting biosynthetic activity.
Triol polyketide synthase (TPKS) is a multifunctional protein with at least four activities as evidenced by the product of the enzymatic activity (Moore, supra). TPKS is believed to be the rate-limiting enzymatic activity(ies) in the biosynthesis of the HMG-CoA reductase inhibitor compounds.
The present invention identifies a DNA encoding triol polyketide synthase (TPKS) from Aspergillus terreus. The DNA encoding the TPKS of the present invention has been isolated, purified and sequenced. Complementary DNA (cDNA) and genomic DNA sequences corresponding to TPKS have been prepared. The TPKS cDNA of the present invention may be used to increase the production of HMG-CoA reductase inhibitors by HMG-CoA reductase inhibitor-producing microorganisms. The TPKS cDNA of the present invention may also be used to produce purified TPKS.